Summary
This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotting, 2-DE or electron microscopy.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Voet, D. and Voet, J. G. (1995) Biochemistry, J. Wiley & Sons, Inc.
Reichert, A. S. and Neupert, W. (2004) Mitochondriomics or what makes us breathe. Trends Genet. 20, 555–562.
Prokisch, H., Andreoli, C., Ahting, U., Heiss, K., Ruepp, A., Scharfe, C., et al. (2006) MitoP2: the mitochondrial proteome database–now including mouse data. Nucleic Acids Res. 34, D705–D711.
Prokisch, H., Scharfe, C., Camp, D. G., II, Xiao, W., David, L., Andreoli, C., et al. (2004) Integrative analysis of the mitochondrial proteome in yeast. PLoS Biol. 2, e160.
Herrmann, J. M., Fölsch, H., Neupert, W., and Stuart, R. A. (1994) Isolation of yeast mitochondria and study of mitochondrial protein translation. In Cell Biology: A Laboratory Handbook (Celis, D. E., ed.), Academic Press, San Diego. Vol. 1, pp. 538–544.
Meisinger, C., Sommer, T., and Pfanner, N. (2000) Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342.
Zischka, H., Weber, G., Weber, P. J., Posch, A., Braun, R. J., Buhringer, D., et al. (2003) Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3, 906–916.
Hannig, K. and Wrba, H. (1964) Isolation of vital tumor cells by carrier-free electrophoresis. Z. Naturforsch. 19, 860.
Hannig, K. and Heidrich, H. G. (1990) Free-Flow Electrophoresis. GIT Verlag, Darmstadt.
Ericson, I. (1974) Determination of the isoelectric point of rat liver mitochondria by cross-partition. Biochim. Biophys. Acta 356, 100–107.
Lewis, P. R. and Shute, C. C. (1969) An electron-microscopic study of cholinesterase distribution in the rat adrenal medulla. J. Microsc. 89, 1811051 –193.
Acknowledgments
The authors thank Dr. E. E. Rojo for critical reading of the manuscript. We also thank Prof. Dr. A. Borst and D. Büringer for their support with electron microscopy. This project was funded by the German Federal Ministry for Education and Research (BMBF) (FKZ: 031U208E, subproject 3; NGFN2 SMP Proteomics, FKZ: 01GR0449, subproject 9, ‘Human Brain’) and the European Union by EU grant LSHG-CT-2003-50520 (INTERACTION PROTEOME).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zischka, H., Kinkl, N., Braun, R.J., Ueffing, M. (2008). Purification of Saccharomyces cerevisiae Mitochondria by Zone Electrophoresis in a Free Flow Device. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-028-7_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-779-2
Online ISBN: 978-1-59745-028-7
eBook Packages: Springer Protocols